Areas of high-density tumor were identified, and 0.6 mm punches were taken from both tumor and adjacent non-tumor prostatic tissue for RNA extraction. The cores were deparaffinized using 800 μL Citrisolv (Fisher Scientific, Pittsburgh, PA) at 60°C for 20 minutes followed by 1.2 mL Citrisolv:absolute alcohol (2:1) at room temperature for 10 minutes. Cores were subsequently washed with absolute alcohol, dried at 55°C, and incubated overnight at 45°C in 300 μL lysis buffer (10 mM NaCl, 500 mM Tris [pH 7.6], 20 mM EDTA, 1% sodium dodecyl sulfate) containing 1 mg/mL proteinase K (Ambion, Austin, TX). RNA was extracted using the RecoverAll Total Nucleic Acid Isolation kit (Ambion). Post tissue digestion, following the manufacturer’s protocol, samples were incubated in 10x DNase (Ambion) and column purified to elute RNA.
Label
biotin
Label protocol
WT-Ovation FFPE System V2 (NuGEN Inc; San Carlos, CA)
Hybridization protocol
Affymetrix protocol
Scan protocol
Affymetrix protocol
Data processing
The data were RMA normalized using Bioconductor package 'oligo' version 1.24.2.
